Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding

Abstract

Bacterial multidrug transporter DrrAB exhibits overlapping substrate specificity with mammalian Pglycoprotein. DrrA hydrolyzes ATP, and the energy is transduced to carrier DrrB resulting in export of drugs. Previous studies suggested that DrrB contains a large and flexible drug-binding pocket made of aromatic residues contributed by several transmembrane helices with different drugs binding to both specific and shared residues in this pocket. However, direct binding of drugs to DrrAB or the mechanism of substrate-induced conformational changes between DrrA and DrrB has so far not been investigated. We used two fluorescence-based approaches to determine substrate binding to purified DrrAB. Our analysis shows that DrrB binds drugs with variable affinities and contains multiple drug binding sites. This work also provides evidence for two asymmetric nucleotide binding sites in DrrA with strikingly different binding affinities. Using targeted fluorescence labeling, we provide clear evidence of long-range conformational changes occurring between DrrA and DrrB. It is proposed that the transduction pathway from the nucleotide-binding DrrA subunit to the substrate binding DrrB subunit includes Q-loop and CREEM motifs in DrrA and EAA-like motif in DrrB. This study lays a solid groundwork for examining roles of various conserved regions of DrrA and DrrB in transduction of conformational changes.

Biochemical characterization of the mouse ABCF3 protein, a partner of the flavivirus-resistance protein OAS1B

“…To determine whether TNP-ATP binds to the ATP-binding pocket(s), increasing concentrations of different nucleotides, including ATP, ADP, or AMP, were added to TNP-ATP-bound ABCF3. It was expected that the addition of nucleotides would displace TNP-ATP from the binding pocket and result in a decrease in fluorescence, as reported previously (44,(55)(56)(57). The addition of 0.1 mM ATP resulted in a sharp decrease in fluorescence indicating displacement of TNP-ATP ( Fig.…”

“…To further investigate nucleotide binding, TNP-ATP, a fluorescent analog of ATP, was used. TNP-ATP alone exhibits some fluorescence in solution; however, its interaction with the nucleotide-binding pocket of a protein results in enhanced fluorescence (44,(55)(56)(57). TNP-ATP binding to ABCF3 resulted in a 2-fold increase in fluorescence (in the presence or absence of 10 mM MgCl 2 ) compared with TNP-ATP in buffer (Fig.…”

“…5, C and D). Because TNP-ATP binding normally occurs with a much higher affinity than ATP binding (44,55,56), the simplest explanation for these data may be that sphingosine can enhance TNP-ATP binding to pocket 2 despite the presence of the K531A mutation, but it is unable to restore its catalytic function. Overall, these data indicate the importance of an intact pocket 2 in sphingosine-stimulated ATP binding and catalysis by ABCF3.…”

“…The ATPase activity of 5 g of purified WT or mutant ABCF3 protein was determined using an ATPase activity assay, as described previously (44,87). The slope of the reaction was measured between 200 and 400 s and used to determine the ATPase activity in nanomoles/min/mg.…”

“…The substrates of the ABCF3 protein are currently not known. However, many ABC family proteins are known to transport lipids and amphiphilic drugs, and their ATPase activities have been shown to be stimulated or inhibited by these substrates (41)(42)(43)(44)(45)(46)(47)(48)(49). Moreover, flavivirus infections modulate host cell lipid metabolism (50) and result in changes in the levels of fatty acids, phospholipids, sphingolipids, and cholesterol in cell membranes (51)(52)(53), including in the ER, which is the site of OAS1B/ABCF3 localization and a major site for lipid biosynthesis (9,54).…”